AN INVESTIGATION TO OBSERVE THE EFFECT OF DMSO

AND GLYCEROL ON THE AGGREGATION OF LYSOZYME

Thesis submitted in partial fulfilment of the requirements for the

degree of
BACHELOR IN TECHNOLOGY
in
BIOTECHNOLOGY
by
Amlan Kumar Sahoo
(110BT0034)
Under the guidance of
Dr. Subhankar Paul

Department of Biotechnology and Medical Engineering

National Institute of Technology
Rourkela
2013-14
 NATIONAL INSTITUTE OF TECHNOLOGY
ROURKELA

CERTIFICATE

This is to certify that the thesis entitled “An investigation to observe the effect of DMSO
and Glycerol on the aggregation of Lysozyme” by AMLAN KUMAR SAHOO
(110BT0034) submitted to National Institute of Technology, Rourkela for the degree of
Bachelor in Technology is a record of bonafide research work, has been carried out by him in
the Department of Biotechnology and Medical Engineering under my supervision and
guidance. To the best of my knowledge, the matter embodied in the thesis has not been
submitted to any other university/institute for the award of any degree or diploma.

Date: Dr. Subhankar Paul

Place: Associate Professor
Dept. of Biotechnology and Medical Engg.
National Institute of Technology, Rourkela.
 ACKNOWLEDGEMENT

I am greatly indebted to, who either through guidance, discussion or providing facilities for the
thesis work, have served as a beacon light or crowned my efforts with success. With an
overwhelming sense of pride and genuine obligation, I express my deep sense of heartfelt
gratitude and regards to my supervisor Dr. Subhankar Paul, Associate Professor of
Department of Biotechnology and Medical Engineering, NIT Rourkela, for his esteemed
guidance and noble supervision during the materialization of this work. I would also like to
thank all faculty members and staff of the Department of Biotechnology and Medical
Engineering, N.I.T. Rourkela for their extreme help throughout the course of my project.

It was an honour to have worked under guidance Ms. Kanti Yadav, Mr. Sailendra Mahanta
and Mr. Deependra K. Ban and I thank them for their constant support and advice in the work
performed in the laboratories for the completion of this project. Lastly, I would like to say that
it was a delight to have worked besides my friend Avishek Parihary and thank him for all the
support and enthusiasm that he has provided me with, without which this project would have
been impossible - we have been through tough times together, but you were sportive enough
to put aside all the plethora of muddles behind and rightfully concentrate and thus gradually
complete the project work.

Amlan Kumar Sahoo

Roll no. –110BT0034
Dept. of Biotechnology and Medical Engg.
NIT Rourkela
 CONTENTS

ABBREVIATIONS iii

LIST OF FIGURES iv

LIST OF TABLES v

ABSTRACT vi

Chapter 1. INTRODUCTION 1

1.1.Introduction 2
1.2.Objective 6

Chapter 2. LITERATURE REVIEW 7

2.1. Lysozyme 8
2.2. Study of molecules preventing Lysozyme aggregation
2.2.1. Acrinidine derivatives prevent lysozyme aggregation 9
2.2.2. Amidated amino acids prevent heat induced aggregation of lysozyme 11
2.2.3. Small molecules like p-Aminophenol and 2-Amino 4-chlorophenol
act as inhibitors for Lys aggregation 12

Chapter 3. MATERIALS AND METHODS 14

3.1.Chemicals Used 15
3.2.Instruments Used 15
3.3.Methodology
3.3.1. Preparation of stock Lysozyme solution 15
3.3.2. Protocols Used for Lysozyme aggregation 15
3.3.2. Protocol used for studying the effect of DMSO on Lys aggregation 16
3.3.3. Protocol used for studying the effect of Glycerol on Lys aggregation 16
3.3.4. Thioflavin T (ThT) Assay 16
3.3.5. Congo Red (CR) Assay 17

i
Chapter 4. RESULTS AND DISCUSSION 18

4.1.Protocol I for Lysozyme aggregation 19

4.2.Protocol II for Lysozyme aggregation 20
4.3.Protocol III (Novel Protocol) for Lysozyme aggregation 21
4.4.Effect of DMSO on Lysozyme aggregation 23
4.5.Effect of Gly on Lysozyme aggregation 24

Chapter 5. CONCLUSION 26

5.1. Conclusion 27

REFERENCES 28

ii
 ABBREVIATIONS

1. ThT – Thioflavin T
2. CR – Congo Red
3. DMSO – Di-methyl Sulphoxide
4. Gly – Glycerol
5. Lys – Lysozyme
6. AA – Amino Acid
7. HSP – Heat Shock Proteins
8. ATP – Adenosine Triphosphate
9. XRD – X-Ray Diffraction
10. H – Bond – Hydrogen Bond
11. UPP – Ubiquitin Proteasome Pathway
12. SDS – PAGE – Sodium Dodecyl Sulphate – Poly Acrylamide Gel Electrophoresis
13. FFF – Field Flow Fractionation
14. CD – Circular Dichroism
15. IR – Infrared
16. NMR – Nuclear Magnetic Resonance
17. HEWL – Hen Egg White Lysozyme
18. GdnHcl – Guanidine Hydrochloride

iii
 LIST OF FIGURES

Sl. No. Figure Title Figure Description Page No.

Figure 1 Graph of results of ThT assay performed on Lys aggregates
1 formed by Protocol I. 19
Figure 2 Graph of results of ThT assay performed on Lys aggregates
2 formed by Protocol II. 20
Figure 3 Graph of results of CR assay performed on Lys aggregates
3 formed by Protocol II. 21
Figure 4 Graph of results of ThT assay performed on Lys aggregates
4 formed by Protocol III. 22
Figure 5 Graph of results of CR assay performed on Lys aggregates
5 formed by Protocol III. 22
6 Figure 6 ThT Assay for effect of DMSO on Lys aggregates 23
7 Figure 7 CR Assay for effect of DMSO on Lys aggregates 24
8 Figure 8 ThT Assay for effect of Gly on Lys aggregates 25
9 Figure 9 CR Assay for effect of Gly on Lys aggregates 25

iv
 LIST OF TABLES

Sl. No. Table Title Table Description Page No.

1 Table 1 Methods used for analysis of protein aggregates 5
2 Table 2 Reagents affecting Lys aggregation 9
3 Table 3 Contents of various test tubes in ThT Assay 17
4 Table 4 Contents of test tubes in CR Assay 17

v
 ABSTRACT

Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, Prions Disease are few among
many diseases caused due to the aggregation of misfolded proteins, which eventually leads to
the formation of amyloids. Amyloids contain a large amount of β – sheets which make it highly
stable in the body environment, thus making its lysis difficult. It is reported that the misfolded
proteins form amorphous aggregates first which contain less number of β – sheets. These
aggregates would further form amyloids as the number β – sheets increase. Inhibiting the
formation of aggregates can be regarded as a therapeutic approach in the treatment of above
mentioned diseases.

In the present investigation, two existing protocols for the formation of amorphous aggregates
of lysozyme (Lys) were studied and a novel protocol for the same was proposed. This novel
protocol showed high amounts of aggregate formation of lysozyme under laboratory
conditions, as observed under Thioflavin T (ThT) assay. However, there was no formation of
amyloids, as was observed under Congo Red assay. The effect of DMSO and Glycerol was
investigated on the formation of Lysozyme aggregate. The results of these assays indicated that
there was significant decrease in the amount of aggregation of lysozyme in solution. Thus, it
was concluded that DMSO and Glycerol act as inhibitors for lysozyme aggregation.

vi
 CHAPTER 1

INTRODUCTION

1|Page
1.1. Introduction:
There are three structural regimes of proteins, namely, primary structure, secondary structure,
tertiary structure and quaternary structure of proteins. Primary structure of a protein refers to
the sequence of amino acids that join together to form the polypeptide chain.

There are several regions in the primary structure which are folded in a particular way. These
structures are called secondary structure. This structure is more regular and is stabilised by
hydrogen bonds between the polypeptide chains. There are several secondary structures like α
– helix, β – sheets etc. The protein is arranged in a coiled shape in α – helix structure. The “α”
means if one looks down the spring, the coiling takes place in a clockwise manner. In the β –
sheet structure, the sheets remain adjacent to each other by hydrogen bonding. The sheets lie
anti – parallel to each other.

Tertiary Structure of protein is more complicated than the previous two structure. Here, the
secondary structure folds itself into a three dimensional structure. This structure is stabilised
by hydrogen bonding, van der waal’s forces and sulphide bridges.

1.1.1. Protein Folding:

A protein exhibits a proper three dimensional conformation called the native structure.
A protein is functional only in its native structure. A loss in the native structure of
protein may result in the malfunctioning of the protein or its inactivation. A protein
achieves this native structure via a series of guided pathways which help the peptide
chains to fold in a desirable way (Lansbury 1999; Merlini and Bellotti 2003; Stefani
and Dobson 2003; Sipe 2005). Any deviation from this pathway can result in misfolded
proteins which may or may not be toxic.
Protein folding takes place under very vulnerable conditions, a change in which can
cause a protein to misfold. This process is assisted by molecules called chaperones.
Chaperones guide the protein folding process towards a pathway that causes the
proteins to fold properly and attain its native state. Once a protein is misfolded, it could
suffer three fates. It could be refolded back to the native state by special molecules
called Heat Shock Proteins (HSP), or it could be lysed by the cell, or it could form
aggregates by hydrophobic – hydrophobic interaction. These aggregates further form
amyloids which are toxic and are the cause of several neurodegenerative disorders like

2|Page
 Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, Prions Disease, Fatal
Filial Insomnia etc.
1.1.2. Protein Aggregation:

It is a biological phenomenon in which unfolded proteins aggregate either intra or extra

cellularly. These aggregates often act toxically. The diseases that occur due to amyloids
are called amyloidosis. After synthesis, a particular state/conformation is obtained
which is called the native state. They are functional only in this state. This folding
process is guided by the hydrophobic forces. However, proteins that are newly
synthesized may not fold correctly or properly folded proteins can misfold
spontaneously. In this case, if the cell doesn’t assist the protein in refolding, or degrade
the misfolded protein, the misfolded proteins form aggregates by interaction between
the hydrophobic cores which become exposed to the surface of the protein when they
misfold (Bennett 2005; Lee et al. 2001; Goedert and Spillantini 2006; Nguyen et al.
1995). There are several causes of protein misfolding. Firstly, mutations in individual
proteins may encode proteins that are particularly prone to misfolding. Secondly,
disruption of pathways to refold proteins (Ubiquitin Proteasome Pathway or UPP) or to
degrade misfolded proteins.
A protein may undergo various aggregation pathways depending upon the
environmental conditions. The aggregate process generally leads to soluble and/or
insoluble aggregates which may precipitate.

1.1.3. Amyloids:

Amyloids, by definition, are insoluble fibrous protein aggregates sharing specific

structural and functional properties. These structures of misfolded proteins alter their
native configuration such that they erratically interact with one another and other
components of the cell, thus forming fibrils that are insoluble. They are responsible for
more than 20 human pathological and neurodegenerative disorders.

A more classical definition of amyloid is that it is an extra cellular proteinaceous deposit

exhibiting β – sheet structure, which are generally identified by apple green
birefringence when stained with Congo Red when observed under polarised light. A
more accurate definition states that it includes any polypeptides which polymerises to
from a cross β – sheet structure in – vivo or in – vitro. Formation of amyloids has been
known to cause many disease like Alzheimer’s disease, Parkinson’s disease,

3|Page
 Huntington’s disease, Fatal Familial Insomnia etc. Amyloid has several distinct
characteristics and structural properties. It has an unbranched structure and ranges from
60 Ao – 120 Ao in diameter (Klunk et al. 1989). It possesses a particular cross X-Ray
Diffraction (XRD) pattern. Amyloid fibrils are formed by proteins that have vastly
different native topologies. Asparagine and Glutamine are commonly found in
amyloids (Chiti et al. 1999; Fandrich et al. 2001). β – sheets assemble through the
formation of H – bonds between adjacent segments of the polypeptide chains. They are
either parallel or anti – parallel. The β – sheets stack parallel to the axis of the fibre with
individual strands perpendicular axis.

1.1.4. Disaggregation of proteins:

Disaggregation of proteins is caused by a special class of molecules called the HSP

(Heat Shock Proteins). All families and sub families of HSP have a common
biochemical function – providing ATP to assist changes in protein folding and their
assembly. HSP 100 plays a very crucial role in reversible refolding of proteins. It has
further been classified into two more sub families - Class 1 HSP 100 (A, B, C, D sub
types) and Class II HSP 100 (M, N, X, Y sub types).

Protein refolding can be achieved in two different ways. Firstly, the misfolded proteins
can be refolded into native structure with the help of HSP molecules. Secondly, external
molecules can be used to achieve the native structure of protein from its misfolded
structure.

1.1.5. Induction factors causing protein aggregation:

Increase in temperature results in increased aggregation. Freezing and Thawing causes

proteins to aggregate. Agitation Stress also is directly proportional to the formation of
aggregates. Protein Concentration is also directly proportional to protein aggregation

4|Page
1.1.6. Methods used for analysis of protein aggregates:
Table 1. Methods used for analysis of protein aggregates.

Method Application
Size estimation and to distinguish from
SDS - PAGE reducible covalent from non – covalent
aggregates.
Capillary Electrophoresis Size estimation and quantification (Soluble
FFF Aggregates)
Static Light Scattering Size and shape estimation
Dynamic Light Scattering Size distribution
Analytical Ultracentrifugation Size, shape estimation and quantification
Coulter Counter Size and member estimation
CD
Fluorescence Spectroscopy Structural Analysis
IR Spectroscopy
NMR Resonance

1.1.7. Lumry – Eyring Two State Model:

This is a model for protein aggregation. According to this model, the native protein
undergoes first a reversible conformational change to an aggregation prone state, which
subsequently assembles irreversibly to the aggregated state. The reversible
conformational change is termed as ‘amorphous aggregate phase’ and the irreversible
aggregated stage is termed as ‘amyloid’. Amorphous aggregates may or may not be
toxic but presence of amyloids in the body is definitely harmful and fatal.

5|Page
1.2. Objectives:

 To study the aggregation process of Lysozyme in vitro with an aim to develop a new
protocol.
 To study the effect of DMSO and Glycerol on the aggregation of Lysozyme.

6|Page
 CHAPTER 2

LITERATURE
REVIEW

7|Page
2.1. Lysozyme:

Lysozyme is a single chain polypeptide of 129 AA cross-linked with four disulphide bridges.
It hydrolyses β(1 – 4) linkages between N-acetylmuraminic acid and N-acetyl-D-glucosamine
residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin. The
enzyme is often used for lysing bacterial cells by hydrolysing the peptidoglycan present in the
cell walls.
This lysozyme preparation is purified from chicken egg white, crystallized three times,
dialyzed, and supplied as a lyophilized powder. Protein content by UV absorbance is 90% with
the remainder (~10%) being buffer salts such as sodium acetate and sodium chloride. The
lysozyme thus prepared is called HEWL. Molecular weight of lysozyme is 14307 Da, making
it easier to study due to its low molecular weight. Lysozyme functions optimally over a pH
range of 6 – 9. The maximum activity of lysozyme is observed at a pH of 6.2. Its isoelectric
point is 11.35.
Inhibitors:
Indole derivatives inhibit Lys aggregation, which bind to and disorient the active site, and
imidazole, which forms a charge-transfer complex. It is also inhibited by surface-active agents
such as sodium dodecyl sulphate, sodium dodecanate, and dodecyl alcohol. Other compounds
having carbon chains of 12 or more carbons in length will also inhibit Lys. Its aggregation is
also inhibited by DMSO and Glycerol as explained in this investigation.
Substrates:
The natural substrate for lysozyme is the peptidoglycan layer of bacterial cell walls. Low
molecular mass substrates including murein degradation products as well as synthetic
compounds have been used for various photometric, isotopic, and immunological lysozyme
assays.

8|Page
Compounds reported to be affecting Lys aggregation:

Table 2. Reagents affecting Lys aggregation.

Sl.No. Compounds that inhibit aggregation Compounds that accelerate
of Lys aggregation of Lys
1 Acrinidine Derivatives Glass
2 Curcumin Steel
3 Kaempferol Silicon
4 Surfactant Coplymers like
Polyethylene Oxide – Polypropylene Plastic
Oxide – Polyethylene Oxide (PEO –
PPO – PEO)
5 p - Aminophenol Rubber
6 2 – Amino 4 - Chlorophenol Oleic Acid

2.2. Study of molecules preventing Lysozyme aggregation:

2.2.1. Acrinidine Derivatives inhibit Lysozyme aggregation:

Formation of amyloids from their native structure has been recognized as a cause for over
twenty human diseases, as well as Alzheimer’s, Parkinson’s and Huntington’s diseases, sort II
polygenic disease, prion-related transmissible spongiform encephalopathies, and hereditary
disease (Taniguchi et al. 2005). The conversion of a selected protein or protein fragment from
soluble native state into insoluble amyloid fibrils ends up in the formation of protein deposits
during a style of organs and tissues with one predominant protein element that's characteristic
of every disease. Varied proteins are known as forming amyloid in vivo.

Though the proteins take issue in their primary and tertiary structures, furthermore as their size
and performance, the extremely ordered amyloid fibrils shaped from these proteins share
common morphological and histochemical staining properties. The amyloid fibrils possess a
typical cross-β structural motif, having b-strands familiarized perpendicular to the fibre axis,
and that they bind by selection the aromatic dyes metal and ThT. as a result of the
morphological similarities between several fibrils from completely different protein building
blocks, it's been hypothesized that completely different proteins follow similar fibre formation
pathways. However, the precise mechanism of the amyloid aggregation remains not clear. It’s

9|Page
typically accepted that protein aggregation has deadly consequence to completely different cell
varieties suggesting its key role in cell impairment and death.

Recently, there's growing proof that soluble oligomers instead of mature amyloid fibrils is also
the most deadly species in amyloid-related disorders. Though this idea was originally
introduced within the investigation of the neurotoxicity of Ab oligomers and their role within
the pathological process of Alzheimer’s disease, this notion has currently been significantly
expanded to incorporate many different proteins concerned in amyloid diseases. The
explanation why early aggregates square measure additional deadly than mature amyloid fibrils
isn't nevertheless clear. In isolated cells, such toxicity has been shown to result from enhanced
membrane permeability with disruption of membrane integrity and formation of particle
channels, aerophilic stress and freeing of cell physiological state by accumulation of animate
thing amyloid. Additional recently, it's been found that amyloid chemical action isn't solely
potential with disease-associated proteins, however conjointly with proteins that don't seem to
be related to any known amyloid disease underneath bound conditions in vitro. Amyloid fibrils
and pre-fibrillar assemblies shaped from non-disease connected proteins have similar
morphological options and toxicity as those detected for disease-associated proteins. This has
light-emitting diode to the suggestion that ability to make amyloid aggregates may be a generic
property of peptide chains, which most or so all peptides and proteins have the potential to
make such structures in vitro underneath applicable conditions. Therefore, the study of the
amyloid aggregation of non-disease associated proteins will increase our understanding of
potential inhibition of amyloid aggregation.

Hereditary general disease is related to one among the simplest known of all proteins-
lysozyme. This disease results from single purpose mutations within the sequence giving rise
to variant proteins that kind huge amyloid deposits within the liver and urinary organ of people
tormented by this disease. Studies of those proteins have shown that amyloid formation of the
variants is as a result of an inclination to favour part denaturated structures. The flexibility to
make amyloid aggregates in vitro has been found for the only purpose mutants and wild-type
human enzymes and conjointly for hen fixings lysozyme. Currently, there aren't any effective
cures for amyloid diseases, however experiments from varied cell and animal models
recommend that the reduction of amyloid aggregation is useful.

The anti-aggregating activity has been known for a spread of gear as well as the antibodies,
artificial peptides, heat shock proteins, and chemical compounds. an excellent range of

10 | P a g e
numerous little molecule compounds are found to inhibit or scale back the aggregation of
varied proteins, notably in relevance Ab deposition, aggregation of enzyme and transthyretin
and also the formation of protease- resistant types of the particle protein. Recently, it's been
detected that anthraquinones square measure ready to inhibit alphabetic character mixture
formation in vitro and in cells. Similar impact was discovered conjointly for different low
relative molecular mass compounds as phenothiazines, N-Phenylamine derivatives,
polyphenols and porphyrins. Acridine based mostly compounds were known as potent
inhibitors of protease-resistant types of the particle protein. Thus, little molecules may offer a
basis for the event of tools for the treatment of amyloid pathology.

2.2.2. Amidated amino acids prevent heat induced aggregation of Lys:

Aggregation is an intrinsic development for peptide chain. The management of aggregation

should be achieved inexpensively and simply for biotechnological and medical applications of
valuable proteins. To scale back aggregation in vitro, numerous factors ought to be tested, such
as pH, ionic strength, temperature, and protein concentration. A straightforward however
effective approach to rising the aggregation drawback is that the addition of low quantity of
potent substance to forestall protein aggregation. Many varieties of additives for reducing
protein aggregation are developed.

Protein-denaturing reagents, usually guanidine and detergents, are used as an aggregation

suppressor that weakens the hydrophobic building block interaction of proteins. However,
these additives ambivalently decrease the soundness of proteins that typically accelerates
aggregation. A compound synthesized through refolding in detergent followed by
cycroamylose addition has been developed to operate as a man-made chaperone. Though non-
denaturing reagents, like amino acids, are accustomed preserve protein answer, their use isn't
adequate to unravel the issues of protein aggregation of these amino acids, essential amino acid
(Arg) possesses a favourable property as an additive for the interference and dissolvation of
aggregation; that's, it doesn't destabilize the native structure and has solely a minor impact on
protein stability whereas it enhancing the solubility of aggregation-prone molecules throughout
refolding.

Recently, we've rumoured that polyamines, specifically spermine and spermidine, forestall the
heat-induced inactivation and aggregation of muramidase a lot of effectively than Arg.
Polyamines slightly destabilize the native structure of muramidase however it markedly will
increase the solubility of aggregation-prone molecules. The indispensable feature within the

11 | P a g e
structure of polyamines for his or her operate as an aggregation suppressor is that the presence
of multiple amines. Essential amino acid ethylester (ArgEE) could be a lot of favourable
additive for suppressing the heat-induced aggregation of muramidase than Arg. though Arg
isn't effective at concentrations below one M, ArgEE is effective at concentrations one order
of magnitude below that of Arg (Vieira et al. 2007; Guijarro et al. 1998). What is more, many
organic compound derivatives equally forestall the heat-induced aggregation of muramidase as
effective as ArgEE. Though organic compound alkylesters are promising candidate for
preventing protein aggregation, these additives could also be hydrolysed to alcohols and amino
acids in a solution. Therefore, organic compound alkylesters don't seem to be favourable for
sensible applications that entail long-time storage.

2.2.3. Small molecules like p-Aminophenol and 2-Amino 4-chlorophenol act

as inhibitors for Lys aggregation:

The conversion of soluble proteins or peptides into amyloid aggregates and their deposition in
tissues are related to variety of necessary human pathologies, together with Alzheimer’s and
Parkinson’s diseases, transmissible spongiform encephalopathies, type-2 polygenic disorder,
and numerous general amyloidoses. Despite the dearth of sequence similarity between distinct
disease-related amyloid-forming proteins and peptides, the method of aggregation looks
similar all told cases, and therefore the ensuing materials share common tinctorial and
morphological characteristics. Additionally, recent studies have shown that the power to create
amyloid aggregates in vitro isn't associate degree exclusive property of proteins associate
degreed peptides related to illness however rather looks to be an intrinsic characteristic of
peptide chains.
Human muramidase variants are involved in chromosome hereditary general diseases. All told
cases studied to this point, single aminoalkanoic acid mutations within the muramidase factor
are shown to steer to the destabilization of the variants associate degreed to an accumulated
propensity to create amyloid aggregates. However, beneath applicable in vitro conditions, wild-
type human muramidase additionally forms amyloid aggregates that are nearly
indistinguishable from those fashioned by the variants (Vernaglia et al. 2004). What is more,
hen ingredient muramidase (HEWL), a protein that's not related to any illness, has recently
been shown to bear amyloid aggregation in vitro at acidic pH and high temperatures. Those
conditions destabilize the native structure of HEWL, pro the population of aggregation-prone

12 | P a g e
partly unpleated species. Muramidase is definitely obtained from completely different sources
and its structure and folding mechanisms are extensively characterised.
These options build muramidase a stimulating model for the study of amyloid aggregation.
Amyloid aggregates of varied proteins are deadly to completely different cell varieties,
however the precise mechanisms of toxicity haven't been totally elucidated. It’s clear, however,
that protein aggregation into soluble oligomers, protofibrils, mature amyloid fibrils, or a mix
of those may be a demand for toxicity. Thus, inhibition or reversion of amyloid aggregation
could represent an attainable therapeutic strategy for the hindrance and treatment of
amyloidosis.

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 CHAPTER 3

MATERIALS AND
METHODS

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3.1. Chemicals Used:
1. Lysozyme
2. Monobasic Sodium Phosphate – Na2HPO4
3. Dibasic Sodium Phosphate - NaH2PO4
4. GdnHCl
5. ThT
6. CR
7. Distilled Water
3.2. Instruments Used:
1. UV/visible spectrophotometer for Congo Red (CR) assay.
2. Fluorescence spectrophotometer for Thioflavin T (ThT) assay.
3.3. Methodology:
3.3.1. Preparation of stock Lys solution:
1 mL of 100 μM Sodium Phosphate buffer was transferred to a test tube from the
stock solution and was diluted to 2 mL with distilled water. 2 mg of Lys was weighed
and was transferred to the test tube containing the buffer. Thus a 1 mg/mL
concentrated Lys solution was prepared before every experiment for use in
aggregation and further while performing ThT and CR assays.

3.3.2. Protocols used for Lys aggregation:

3.3.2.1. Protocol I:
The stock buffer was diluted to 20 mM and its pH was adjusted to 6.3±0.1. A 10 μM
solution of lysozyme was prepared. It was transferred into a test tube along with 3M
GdnHCl. The solution was transferred into a small beaker and was kept on a
magnetic stirrer which was maintained at 60 rpm at 50 oC.
3.3.2.2. Protocol II:
A solution containing 1 mg/mL Lys and 50mM Sodium Phosphate Buffer was
prepared and the pH was adjusted to 7.16. 200 μL aliquot of the stock solution was
taken in a microtube and was then placed in a water bath. Temperature was increased
from 25 oC to 90 oC at 1 oC/sec. It was stored at 25 oC for 20 min. It was then
centrifuged at 15000 g for 20 min at 25 oC.

15 | P a g e
 3.3.2.3. Protocol III (Novel Protocol):
A stock solution of 1 mg/mL Lys was prepared in 50 mM sodium phosphate buffer
and the pH was adjusted to 7.16. 200 μL of the stock solution was taken in a test tube
and the volume was adjusted to 1 mL using the buffer. It was then heated at 100 oC
for 20 min in a water bath. It was incubated at 25 oC for 20 more min after heating.

3.3.3. Protocol used for studying the effect of DMSO on Lys aggregation:
Two test tubes were taken and were marked “BB” and “AB”. 1 mL of 1 mg/mL Lys
stock solution was taken in both test tubes. 100 μL of DMSO having volume fraction
= 1 (pure DMSO) was added to the test tube marked “BB”. Both the test tubes were
closed using cotton plugs and aluminium foil. They were kept in the water bath for
20 min at 100 oC. After heating, both the test tubes were stored for 20 more min at
room temperature to allow the DMSO to act properly on the aggregates formed in
the test tube marked “BB” after boiling.

3.3.4. Protocol used for studying the effect of Glycerol on Lysozyme

aggregation:
Two test tubes were taken and were marked “BB” and “AB”. 1 mL of 1 mg/mL Lys
stock solution was taken in both test tubes. 100 μL of Glycerol having volume
fraction = 1 (pure Glycerol) was added to the test tube marked “BB”. Both the test
tubes were closed using cotton plugs and aluminium foil. They were kept in the water
bath for 20 min at 100 oC. After heating, both the test tubes were stored for 20 more
min at room temperature to allow the Glycerol to act properly on the aggregates
formed in the test tube marked “BB” after boiling.
3.3.5. Thioflavin T (ThT) assay:
A 5 mM solution of ThT dye was prepared in sodium phosphate buffer of pH 7.16.
This dye was stored in a dark and cold place for further use during ThT assay.
Four test tubes were taken and were marked “C” (Control Solution), “A”
(Aggregated Solution), “BB1” (Addition of reagent before boiling), and “AB1”
(Addition of reagent after boiling). The contents of the test tubes are portrayed in
Table 3. All the test tubes were then left in the dark for 30 min before performing
the assay because ThT dye is a light sensitive.

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 Table 3. Contents of the various test tubes in ThT Assay
Test Tube Lysozyme Reagent ThT Buffer Total Vol.
C Non – aggregated stock
(100 μL) - 15 μL 2885 μL 3 mL
A Aggregated solution
from test tube “AB” - 15 μL 2885 μL 3 mL
(100 μL)
AB1 Solution from test tube
“AB” (100 μL) DMSO/Gly 15 μL 2785 μL 3 mL
BB1 Solution from test tube
“BB” (200 μL) DMSO/Gly 15 μL 2785 μL 3 mL

3.3.6. Congo Red (CR) assay:

7mg/mL of CR solution was prepared in sodium phosphate buffer at pH = 7.16. This
dye was stored in a dark and cold place for further use during CR assay.
Five test tubes were taken and were marked “CR” (Congo Red), “C” (Control Solution),
“A” (Aggregated Solution), “BB1” (Addition of reagent before boiling), and “AB1”
(Addition of reagent after boiling). The contents of the test tubes are portrayed in the
following table. All the test tubes were then left in the dark for 30 min before performing
the assay because CR dye is a light sensitive.

Table 4. Contents of the various test tubes in Congo Red (CR) Assay
Test Tube Lysozyme Reagent CR Buffer Total Vol.
CR - - 15 μL 2985 μL 3 mL
C Non – aggregated stock
(100 μL) - 15 μL 2885 μL 3 mL
A Aggregated solution
from test tube “AB” - 15 μL 2885 μL 3 mL
(100 μL)
AB1 Solution from test tube
“AB” (100 μL) DMSO/Gly 15 μL 2785 μL 3 mL
BB1 Solution from test tube
“BB” (200 μL) DMSO/Gly 15 μL 2785 μL 3 mL

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 CHAPTER 4

RESULTS AND
DISCUSSION

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4.1. Protocol I for Lysozyme (Lys) aggregation:

Here, the protocol was used to test for Lys aggregation and was tested after 0h, 15h and 24 h
for the amount of aggregation using the ThT assay. As expected, the buffer showed the least
intensity. It was subtracted from the graphs of all the solutions to obtain accurate results without
zero error. Although the aggregation in the sample was observed to gradually increase with
escalating time, a major anomaly in the results were observed. The graph for the “Control”
solution which contained 1mg/mL non-aggregated Lys solution in sodium phosphate buffer
(pH=6.4) was observed to have suffered more aggregation that the samples which were
prepared using this protocol and were tested after 0 h and 15 h, which is an anomaly considering
the fact that the control solution contains “non-aggregated” Lys. This anomaly was attributed
to the presence of GdnHCl (Guanidine Hydrochloride) in the solution which is thought to be
the reason of decrease in aggregation in aggregated solutions tested after 0 h and 15 h, though
the clear mechanism has not yet been understood. Another important factor that played a role
in rendering this protocol ineffective is the difference in laboratory conditions.

4.1.1. Thioflavin T (ThT) Assay:

Figure 1. Graph of results of ThT assay performed on Lys aggregates formed by Protocol I.
(1) Lysozyme aggregation observed after 24 h (2) Native lysozyme solution (3) Lysozyme
aggregation observed after 15 h (4) Lysozyme aggregation observed after 0 h (5) Sodium
Phosphate buffer.

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4.2. Protocol II for Lys aggregation:

From the graph of ThT assay, we can observe that the aggregation is less in the test solution
than in the control solution, which is absurd considering that control solution itself contain
“non-aggregated” lysozyme. Moreover, no peaks were observed for control and test solutions
in the CR assay, although a peak was observed for the CR solution. This indicated nothing
about the formation of aggregates. As the results for both the assays in this protocol didn’t
predict desired results, this protocol was deemed inaccurate.

4.2.1. ThT Assay:

Figure 2. Graph of results of ThT assay performed on Lys aggregates formed by Protocol II.
(1) Native lysozyme solution (2) Aggregated lysozyme solution.

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4.2.2. Congo Red (CR) Assay:

Figure 3. Graph of results of CR assay performed on Lys aggregates formed by Protocol II.
(1) Pure Congo Red solution (2) Native Lysozyme solution (3) Aggregated Lysozyme solution.

4.3. Protocol III (Novel Protocol) for Lys aggregation:

As observed from the graph of ThT assay, there was a significant amount of aggregation in the
test solution as compared to the control solution which is ideally termed as “zero aggregation
state”. This is based on a simple mechanism. Heating Lys at 100 oC for 20 min causes the
protein to denature. This makes the hydrophobic cores of the protein to protrude out of the
conformation which were on the inside in its native conformation. The hydrophobic –
hydrophobic interactions causes the protein to aggregate.

There was very minimal difference in peaks and the wavelengths at which peaks were observed
for control and test solution as observed from the graph of CR assay. This indicates that there
was no amyloid formation in Lysozyme.

This protocol was dubbed as accurate and perfect for Lysozyme aggregation under the
laboratory conditions.

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ThT Assay:

Figure 4. Graph of ThT assay performed on Lys aggregates formed by Protocol III.
(1)Aggregated lysozyme solution (2) Native lysozyme solution (3) Sodium Phosphate buffer.

4.3.1. CR Assay:

Figure 5. Graph of Congo Red assay performed on Lys aggregate formed by Protocol III. (1)
Aggregated lysozyme solution (2) Native lysozyme solution (3) Pure Congo Red solution.

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4.4. Effect of DMSO on Lys aggregation:

As observed from the ThT assay, DMSO significantly decreased the aggregation in Lys, both
when added before and after boiling. This again follows a simple mechanism. At high
concentrations of DMSO, the protein attempts to minimise the contact area between DMSO
and amino acid residues for which DMSO is a bad solvent, so that it will rearrange in such a
way so as to expose the units that were buried in native conformation and vice versa. In short,
it causes the protein to flip it structure. Thus, flipping aggregates causes low levels of
hydrophobic – hydrophobic interaction and thus reduces aggregation.

Again, no major peak shifts were observed in the CR assay indicating that there was no amyloid
formation in the solution.

4.4.1. ThT Assay:

Figure 6. ThT Assay for effect of DMSO on Lys aggregation. (1) Aggregated lysozyme
solution (2) DMSO was added to lysozyme solution before boiling (3) DMSO was added to
aggregated solution after boiling (4) Sodium Phosphate buffer (5) Native lysozyme solution.

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4.4.2. CR Assay:

Figure 7. CR Assay for effect of DMSO on Lys aggregates. (1) DMSO was added to Lysozyme
solution before boiling (2) Aggregated lysozyme solution (3) Native lysozyme solution (4)
Pure Congo Red solution (5) DMSO was added to aggregated lysozyme solution after boiling.

4.5. Effect of Glycerol (Gly) on Lys aggregation:

As observed from the ThT assay, Gly significantly decreased the aggregation in Lys, both when
added before and after boiling. This, again follows a simple mechanism. Poly co – solvents like
glycerol increases the thermal stability of Lys. And proteins in general. This is explained by
preferential hydration favouring the more compact native rather than the denatured state.

Again, no major peak shifts were observed in the CR assay indicating that there was no amyloid
formation in the solution.

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4.5.1. ThT Assay:

Figure 8. ThT Assay for effect of Glycerol on Lys aggregates. (1) Aggregated lysozyme
solution (2) Glycerol was added to native lysozyme solution before boiling (3) Glycerol was
added to lysozyme aggregate solution after boiling (4) Sodium Phosphate buffer (5) Native
lysozyme solution.

4.5.2. CR Assay:

Figure 9. CR Assay for effect of Glycerol on lysozyme aggregates. (1) Glycerol was added to
native lysozyme solution before boiling, and then the solution was heated (2) Glycerol was
added to aggregated lysozyme solution after boiling (3) Native lysozyme solution.

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 CHAPTER 5

CONCLUSION

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1.1. Conclusion:

The aggregates formed from the boiling of Lysozyme (Lys) at 100 oC for 20 min were quite
significant. These aggregates are normally called ‘amorphous’ aggregates. Further increase in
the number of β – sheets would result in the formation of amyloids which is the cause of several
neurodegenerative disorders. An attempt was made to stop the amyloid formation pathway at
the ‘amorphous’ aggregate stage and the effect of DMSO and Glycerol (Gly) were observed
by performing ThT and CR spectroscopic assays.

DMSO and Gly were observed to be inhibitors of Lys aggregation. DMSO was found to
prevent aggregation significantly, both when added before and after boiling. Gly also showed
signs of inhibiting Lys aggregation, but was a weak one. Introduction of DMSO into the
aggregated solution caused the proteins to flip their structure inside out due to preferential
solubility of amino acid in DMSO. This reduced the hydrophobic – hydrophobic interactions
amongst proteins and thus decreased aggregation. Gly prevented aggregation due to its
preferential hydration favouring the more compact native structure rather than the denatured
state. Although the mechanisms of action of both the molecules were different, they were found
to be effective inhibitors of Lys aggregation.

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